Our proposed research will focus on the molecular mechanism of antigen recognition by T cell receptors (TCR). TCRs are responsible for recognizing peptide-MHC (pMHC) ligands displayed on the surface of antigen-presenting cells. TCR recognition determines the selection, development, differentiation, fate, and function of a T cell. Thus, understanding the mechanism of TCR antigen recognition is of critical importance in T cell immunology. Antigen recognition by TCRs has three important characteristics: (1) it is governed by a dynamic TCR-pMHC interaction at the live cell membrane. (2) It is very sensitive-a T cell can recognize a single foreign pMHC in the presence of abundant self-pMHCs. (3) It is very specific-TCRs can discriminate between even closely related amino acids in the antigenic peptide and elicit distinct functional responses. This complexity reflects the uniquely demanding nature of TCR recognition, which requires the detection of a very weak `signal' (very rare foreign pMHCs) in the presence of considerable `noise' (abundant self- pMHCs) at the surface of the cell being surveyed. It has motivated intensive research in order to understand the fundamental molecular mechanisms driving different aspects of this process over the past two decades. Many models have been proposed based on the structure, thermodynamic properties, kinetics and signaling of TCR-pMHC interactions, but the molecular mechanism governing TCR engagement with pMHC ligands remains controversial. A fundamental problem of these studies is that they cannot directly visualize and continuously measure the in situ dynamic interactions between TCRs and pMHCs at the live cell membrane. We recently found that TCRs bind to pMHCs with fast kinetics (Huang et al., 2010, Nature) and that a single pMHC triggers the formation of a long-lasting TCR microcluster and subsequent cytokine secretion (Huang et al., 2013, Immunity). Therefore, we hypothesize that multiple TCRs serially engage with a small number of pMHC ligands to accumulate enough stimulatory signal to reach a threshold for T cell triggering. The TCR serial engagement model was initially proposed to explain the observation that a small number of pMHCs can trigger the dowregulation of hundreds of TCRs (Valitutti et al., 1995, Nature). However, this model is incomplete and it lacks direct experimental evidences at the molecular level. We recently engineered a small, stable, specific, blinking-suppressed, monovalent quantum dot (QD). Based on a Cy3/Cy5 based single-molecule Frster resonance energy transfer (smFRET) assay recently developed in the Mark Davis' laboratory (Huppa et al., 2010, Nature), we propose to devise a novel QD-based smFRET to directly visualize and continuously measure serial TCR-pMHC interactions in situ. This will give us a very bright and stable FRET reagent that will enable us to directly measure repeated engagement of a same pMHC with multiple different TCRs at a temporal resolution that should be able to capture the dynamics of these events. The results will allow us to test our central hypothesis and settle a longstanding question in immunology.